New Generation of Clickable Nucleic Acids: Synthesis and Active

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New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA Xun Han, Dylan W. Domaille, Benjamin D. Fairbanks, Liangcan He, Heidi R. Culver, Xinpeng Zhang, Jennifer N. Cha, and Christopher N. Bowman Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01164 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA Xun Han,† Dylan W. Domaille,‡ Benjamin D. Fairbanks,† Liangcan He,† Heidi R. Culver,† Xinpeng Zhang,† Jennifer N. Cha,† Christopher N. Bowman*,† †

Department of Chemical and Biological Engineering, University of Colorado, UCB 596,

Boulder, Colorado, 80309, United States ‡

Department of Chemistry, Colorado School of Mines, 1500 Illinois St., Golden, Colorado,

80401, United States KEYWORDS. click chemistry, nanoparticle, nucleotides, photopolymerization, thiols

ABSTRACT. Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photo-initiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of 6 atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in thirty seconds as

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characterized by size-exclusive chromatography (SEC) and Infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

INTRODUCTION DNA stores the genetic information of living organisms via its simple alphabet of four nucleobases, thymine (T), adenine (A), cytosine (C), and guanine (G). Watson-Crick base pairing enables accurate recognition between two complementary nucleobases through hydrogen bonding, and ensures the high fidelity during replication and transmission of the genetic information. Since the double helix structure of DNA was uncovered in 1953,1 artificial oligonucleotides that are able to readily hybridize with complementary, natural single stranded DNA (ssDNA) have been the focus of significant interest and applications owing to the DNA mimics’ promising applications in molecular biology, genetic diagnostics, pharmaceutics, and biosensors.2 One class of these synthetic oligonucleotides is obtained by modifications of the natural DNA’s sugar ring, such as locked nucleic acid (LNA),3 threose nucleic acid (TNA),4 1’,5’-anhydrohexitol nucleic acid (HNA),5 and glycol nucleic acid (GNA).6 These molecules adopted the phosphodiester backbone to take advantage of prevalent DNA synthesis techniques. In 1991, Nielsen et al. reported a new type of DNA mimic, referred to as peptide nucleic acids (PNAs), which are characterized by a peptidic backbone structure and possess remarkable hybridization attributes with complementary DNA and RNA molecules.7 However, both the phosphoramidite and peptide solid-phase synthesis approaches adopt a sequential and stepwise synthetic strategy, which has significantly limited the synthetic efficiency, production scale, and


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applications of those synthetic DNAs, especially, when the target polynucleotides consist of repetitive sequences. Polymerization of nucleotides is a straightforward strategy for enlarging the synthesis scale and expanding the scope of applications for these uniquely capable molecules. Recently, several common polymerization methods, such as atom transfer radical polymerization (ATRP)8 and ring-opening polymerization (ROP),9 have been applied to synthesize DNA analogues. However, the lack of sequence control and minimal sequence specific interaction with natural oligonucleotides significantly restrict the characteristics and capabilities of these molecules. In 2015, clickable nucleic acids (CNAs) were synthesized by employing a photo-initiated thiol-ene click reaction as the polymerization method, and a sequence-controlled synthesis platform was also designed.10 Benefitting from the power of click chemistry and photopolymerization, CNA synthesis was accomplished with high atom economy via a robust and environmentally friendly method to produce sequence-controlled DNA analogues in short time and in significant scale. Unfortunately, while this CNA demonstrated sequence specific binding with complementary ssDNA, it did so only in contexts where it was highly entropically favored (e.g. on the surface of particles).11 One major reason for this drawback is attributed to the mismatch of the spacer length between the two nucleobases in CNA and DNA (Figure 1). The backbone structures of natural DNA and RNA as well as previous artificial DNAs have generally had repeat units of 6 atoms, while the backbone present in the previously reported CNA has a 7-atom repeat unit. Therefore, we redesigned the structure of CNA and, herein, report the synthesis and characteristics of this second generation CNA (CNA-2G) that conforms to the backbone spacing of native nucleic acids. Notably, comparing to the first generation CNA (CNA-1G), CNA-2G manifests


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significantly higher affinity with complementary ssDNA but also has even greater polymerization efficiency.

Figure 1. Backbone structures of natural DNA, PNA, CNA-1G, and CNA-2G. NB = nucleobase.

EXPERIMENTAL SECTION Materials. All chemical reagents were purchased from Sigma Aldrich or Fisher and used without further purification. DNA (A10 and T10 ssDNAs) was bought from Integrated DNA Technologies, Inc. Cadmium sulfide nanoparticles were bought from TED Pella. Instrumentation. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were collected using a Bruker Advance-III 400 spectrometer. High resolution mass spectra and analytical data were obtained via the PE SCIEX/ABE API QSTAR Pulsar Hybrid LC/MS/MS. Polymer molecular weights were estimated using a TOSOH ECO SEC HLC- 8320 GPC equipped with two polystyrene columns and UV and RI detectors. For these polymers, the UV detector was set at 260 nm and DMSO was used as the eluent at 50 °C. The output data of Mn were calibrated by short first generation of CNA (CNA-1G) oligomers. Photopolymerization was performed with Thorlabs DC4104 4-Channel LED driver. Transmission electron microscopy (TEM) images were obtained from FEI Tecnai T12 Spirit TEM.


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Synthesis of dichloride intermediate 3. To a solution of 2-chloroethylamine hydrochloride (35 g, 0.30 mol) in water (50 mL) was added NaOH (18 g, 0.40 mol) in portions. The resulting solution was extracted with diethyl ether (3X100 mL). The combined organic layers were dried over MgSO4, then added to a 1 L round-bottom flask filled with acetaldehyde (26 g, 0.59 mol), 200 mL diethyl ether, and MgSO4 (40 g, 0.33 mol) at room temperature. The resulting mixture was stirred at room temperature for 30 min, then filtered. The filtrate was concentrated in vacuum and re-dissolved in toluene (300 mL). To a fresh imine toluene solution were added chloroacetyl chloride (20 mL, 0.25 mol) and N,N-diethylaniline (40 mL, 0.25 mol). The resulting brown mixture was stirred at room temperature for 1 hour before being quenched with water. The organic layer was separated and dried over MgSO4, filtered, and concentrated. The residue was purified by flash column chromatography (hexanes:EtOAc = 20:1 to 4:1) in silica gel to afford the desired product (30 g, 66% yield, mixture of two diastereomers, d.r. = 4.0:1) as a pale yellow oil. Synthesis of thymine pendant intermediate 4. To a suspension of thymine (6.9 g, 55 mmol) and compound 3 (10 g, 55 mmol) in CH2Cl2 (500 mL) was added 1,8-diazabicyclo(5.4.0)undec7-ene (9.5 mL, 60 mmol) dropwise. The resulting mixture was stirred at room temperature for 5 hours, then charged silica gel. The solvent was removed under vacuum. A dry-pack flash column chromatography (CH2Cl2:MeOH = 100:0 to 10:1) was set up to produce desired pure nucleotide 4 (6.8 g, 46%, mixture of two diastereomers, d.r. = 2.2:1) as a white powder. Synthesis of thymine pendant intermediate 5. To a solution of compound 4 (1.1 g, 4.0 mmol) in DMF (5 mL) were added potassium thioacetate (1.4 g, 12 mmol) and NaI (30 mg, 0.20 mmol) successively. The resulting mixture was stirred at 55 °C overnight, then the solvent was removed under vacuum. The residue was diluted with CH2Cl2 (20 mL), then charged with silica gel. The


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mixture was dried under vacuum. A dry-pack flash column chromatography (CH2Cl2:MeOH = 100:0 to 10:1) was set up to afford acetyl protected monomer 5, which was further purified through recrystallization from EtOH/water (1/1) to yield pure desired product (914 mg, 63% yield, mixture of two diastereomers, d.r. = 1.7:1) as a light brown powder. Synthesis of thymine CNA-2G monomer 6. To a suspension of compound 5 (330.5 mg, 1.06 mmol) in MeOH (3 mL) was added 4.5 M NaOH aqueous solution (0.5 mL, 2.25 mmol) in one portion. The resulting mixture was stirred at room temperature for 5 min, at which time it turned to a brown clear solution. Then, 1 M HCl aqueous solution was slowly added to neutralize the reaction solution (pH ≈ 7). Meanwhile, precipitates came out and were collected by filtration, then washed with acetone twice to afford pure monomer (246.5 mg, 87% yield, mixture of two diastereomers, d.r. = 1.7:1) as a brown powder. Preparation of CNA-2G polymers 7. To a mixture of monomer 6 (246.5 mg, 0.92 mmol) and DMSO (920 mg) in 3 mL a glass vial was added a stock solution of DMPA (80 mg, 2.85 wt% in DMSO, 0.0092 mmol). The vial was swirled gently until the mixture formed a homogeneous solution. Then, the solution was exposed under LED 365 nm UV light with an intensity of 20 mW/cm2 for 10 minutes. The resulting solution was dropwisely added to acetone (45 mL) in a 50 mL centrifuge tube. The white precipitate was washed with acetone (2 x 45 mL). The precipitate was dried under vacuum to afford polyT CNA-2G (103.5 mg, 42% yield) as a white powder. The precipitate-washed acetone was collected, concentrated, diluted in water, and extracted with CH2Cl2 (3 x 50 mL). The combined organic layers were charged with silica gel and dried under vacuum. The resulting dry silica gel was loaded onto a cartridge and purified by flash chromatography (CH2Cl2:MeOH = 100:0 to 10:1) to afford the cyclic by-product 8 as a white solid.


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Gel electrophoresis experiments. All the nucleotide samples were prepared in 50% DMSO and 50% 0.5 x Tris/Borate/EDTA (TBE) buffer at the desired concentrations and annealed for 45 min from 55 °C to 5 °C prior to loading on 20% Novex TBE gelsl. All the gels were run at 75 V and 0 oC for certain times. Synthesis of CdS-CNA nanoparticles. To a solution of oleic acid stabilized CdS nanoparticles (4.6 mg) in chloroform (3 mL) were added NaOH aqueous solution (2 mL, 50 mM) and mercaptoethanol (50 µL) successively. The resulting mixture was stirred at room temperature overnight, followed by centrifugation in 30K filter. The precipitate was collected and redispersed in DMSO/water (1/1, 0.8 mL) to form a mercaptoethanol-modified CdS nanoparticle stock solution with a nanoparticle concentration of 7.69 µM, which was determined by UV/Vis spectra (absorption peak at 472 nm, extinction coefficient = 2.56 x 106 mol/(L.cm)). To the freshly made mercaptoethanol-modified CdS nanoparticle stock solution (7.69 µM, 30 µL) were added a solution of polyT CNA-2G in DMSO (6.9 µL, 5 mM), a solution of 2K PEG-SH in water (2.3 µL, 5mM), water (4.6 µL), DMSO (5.5 µL), and NaHCO3 aqueous solution (5.5 µL, 500 mM) successively. The resulting solution was stirred at room temperature for 24 hours, followed by centrifugation in 30K filter. The precipitate was collected and re-dispersed in DMSO/water (1/1, 25 µL) to form a CdS-CNA nanoparticle stock solution with a concentration of 7.69 µM. 94 CNA polymers are on each CdS nanoparticle, which was determined by CNA concentration standard curve (Figure S5, Supporting Information). CdS-CNA nanoparticles self-assembly. To a freshly made CdS-CNA nanoparticle stock solution (3.9 µL, 7.69 µM) were added DNA-A5 linker aqueous solution (3.0 µL, 463.3 µM), DNA-complementary linker aqueous solution (3.1 µL, 454.0 µM), DMSO (8.1 µL), and NaCl aqueous solution (2.0 µL, 5 M) successively. The resulting solution was annealed at 60 °C for 10


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min. After annealing, the solution was cooled down to room temperature at a rate of 1 °C/10 min, then cooled down to 4 °C in 1 hour. The resulting solution was kept at 4 °C for further characterization.

RESULTS AND DISCUSSION Synthesis of thymine CNA-2G monomer. To shorten the backbone of CNA-1G by one carbon, a new thiol-ene type monomer was designed, containing a highly reactive vinylamide structure instead of the allylamide or acrylamide skeleton in CNA-1G monomers (Scheme 1).10,11 While the difference in structure is seemingly minor, designing an effective synthetic strategy for this vinylamide monomer proved unexpectedly challenging. Routine amide vinylation protocols, such as elimination12 and metal-catalyzed coupling,13 were unsuccessful. Thus, a new synthetic methodology was developed to assemble the vinylamide in a one-pot and scalable manner from commercially available starting materials. Deprotonation of 2-chloroethylamine hydrochloride generated the unstable amine 1 in situ, which directly condensed with acetaldehyde to yield another labile species, imine 2, with the assistance of desiccant. Following the addition of an electrophile, chloroacetyl chloride, the imine intermediate was immediately captured and went through a double bond rearrangement to afford vinylamide 3 in good yield and a 4/1 ratio of two diastereomers. Then, the ⍺-chloride and alkyl chloride of vinylamide 3 were substituted by nucleobase and thiolacetate successively to give nucleotide precursor 5 that was transformed to target monomer 6 as a mixture of disastereomers with 1.7/1 ratio by a basic deprotection. The synthetic route proceeded via 4 single steps from inexpensive materials and delivered gram-scale CNA-2G monomer in 17% overall yield.


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O

O

.

NH2 HCl

Cl

NaOH

NH2

Cl

Et2O, r.t.

H

, MgSO4 Et2O, r.t.

1

O Cl

N

DCM, r.t.

3, 66%, d.r. = 4/1

N

O

KSAc, NaI

N

1.

The

synthetic

route

N

for

O

NaOH

N

CNA-2G

O

O HS

5, 63%, d.r. = 1.7/1

thymine

N

MeOH, r.t.

O

AcS

4, 46%, d.r. = 2.2/1

Scheme

NH

NH DMF, 55 oC

O Cl

O

O NH

Thymine, DBU

toluene, r.t.

2

O Cl

N

Cl

Cl , PhNEt 2

Cl

N

6, 87%, d.r. = 1.7/1

monomer.

DBU

=

1,8-

diazabicyclo(5,4,0)undec-7-ene, Ac = acetyl. Photopolymerization. With freshly synthesized monomer 6 in hand, photopolymerization was initiated with variable conditions (Table 1). Disulfide formation that limited the desired thiol-ene polymerization was observed under some circumstances if the monomers were not used in this manner. Initially, the reaction was conducted by irradiation with 365 nm light with 5 mol% loading of photoinitiator, 2,2-dimethoxy-2-phenylacetophenone (DMPA), and 1.0 mol/L concentration of 6 in DMSO at room temperature (entry 3). The desired polythymine (polyT) nucleotides 7 were obtained in 46% yield (by 1H NMR) and with 4100 number-average molecular weight (Mn) according to size-exclusive chromatography (SEC) analysis, which is equivalent to a degree of polymerization (DP) of ~15.

Table 1. Photopolymerization of CNA-2G monomer 6 at different conditions.a


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Self-cyclized by-product 8 was also observed in 54% yield (by 1H NMR). A similar by-product with a 7-membered cycle was isolated after photopolymerization for CNA-1G monomer as well in 58% yield,13a which contradictorily suggested that, in the polymerization of 6, the more thermodynamically favored 6-membered by-product would overwhelm the polymeric products. We speculate that it might be due to the different cyclization tendencies of the two diastereomers of monomer 6 (Figure 2). Diastereomer 6a goes through a thermodynamically favored chair-like transition configuration to afford 8, while the disfavored boat-like transition configuration effectively limits the cyclization of 6b. Therefore, cyclization is favored under lower monomer concentration (entries 1-3) because of the reduced possibilities for cyclization-favored 6a to be captured by the propagating chain. Moreover, the loading of photoinitiator does not influence the Mn, PDI, and the polymerization/cyclization ratio significantly (entries 3-5). Each condition of CNA-2G polymerization resulted in polyT 7 with relatively narrow PDI values, which is not feasible from a classic step-growth reaction mechanism but hinted at a more chain-growth-like mechanism. An intramolecular chain-transfer reaction might be involved in both chain growth propagation and cyclization processes. Besides, a control group was arranged to verify the photo-


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initiated mechanism of this polymerization (entry 6). Without photoinitiator, there was no conversion observed by 1H NMR after 10 min irradiation, which indicated that the photoinitiator is necessary for this polymerization, at least on these relatively short timescales, though thiol-ene polymerization has been shown to be effective even without photoinitiators under some conditions.14

Figure 2. Self-cyclization of two diastereomers of monomer 6. DMPA = 2,2-dimethoxy-2phenylacetophenone. Under suitable reaction conditions, photopolymerization of 6 was conducted in a scale of a quarter-gram (entry 5). By-product 8 was conveniently separated by precipitating the crude solution into acetone, which gave the pure polyT as a white powder in 42% isolated yield. The structure of polyT was confirmed by 1H NMR spectroscopy (Figure 3). With sufficient amplification of the 4.5 to 7.5 ppm region, the characteristic peaks of the terminal vinyl group were observed, consistent with the linear structure of polyT CNA-2G.


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Figure 3. 1H NMR of polyT CNA-2G.

The photopolymerization efficiency was investigated by SEC and Fourier Transform Infrared (FTIR) (Figure 4) spectroscopy. The molecular weight evolution given by SEC for irradiation times of 0-600s indicated that the polymerization neared completion in only 30 seconds at room temperature, under 20 mW/cm2 intensity of 365 nm light, and with 1 mol% loading of photoinitiator. Relatively high-molecular-weight polymers (Mn = 3600) were formed within the first ten seconds of light exposure. Increased exposure increased the average molecular weight of the polymers, but by relatively little with respect to increasing conversion. This counter-intuitive observation indicates a chain-growth-like propagation mechanism since the step-growth mechanism would be expected to exhibit a slow initial evolution of molecular weight that increases dramatically at higher conversions. The low molecular-weight peak at 18.2 minutes, sharing an elution time with pure monomer, represents the cyclized byproduct depicted in Figure 2. The disappearing peak at 17.4 minutes was shown to belong to a disulfide-linked dimer that is formed during the SEC separation (See Supporting Information, Figure S1). Furthermore, a “conversion versus time” relationship was obtained from FTIR based on monitoring the amount of remaining thiol-associated peak at 2540 cm-1 with increasing irradiation time. Consistent with the SEC results, with 1 mol% loading of DMPA and 20 mW/cm2 light intensity, nearly


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quantitative thiol group consumption was achieved in less than 30 seconds. Lowering the light intensity to 1 mW/cm2, the polymerization was still completed in 4 minutes. a)

b)

Figure 4. a) Overlaid SEC traces of CNA-2G monomer 6 polymerization at different exposure times. Polymerization was conducted at 1.0 mol/L 6 in DMSO, 1 mol% DMPA, and 20 mW/cm2 365 nm UV light. Samples of each run of the SEC came from the same polymerization at the indicated times. b) Overlaid “conversion vs time” plots of CNA-2G monomer 6 polymerization under different exposure intensities. Polymerization was conducted at 1.0 mol/L 6 in DMSO, 1 mol% DMPA, and 365 nm UV light.

Gel electrophoresis experiments. The ability to bind with high specificity to complementary ssDNA is a vital characteristic of DNA mimics, requisite for virtually any desired application. The interaction between CNA-2G polymers and ssDNA was evaluated by gel electrophoresis (Figure 5). The titration experiment (Figure 5a) indicated a clear hybridization at ratios of polyT CNA-2G/polyA ssDNA greater than 7.5 (Figure 5a, Lane 6). Increasing the relative excess of CNA led to a more completely hybridized fraction of the polyA ssDNA (Figure 5a, Lanes 7-12). The intensity of the unbound DNA band in each lane was quantified by using the gel analysis feature in ImageJ software based on the fluorescence intensity of bands and plotted against poly


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T CNA-2G concentration. The data ware fit to a sigmoidal curve and the Kd of polyT CNA2G/polyA ssDNA interaction was determined under these conditions to be approximately 70 µM (Figure 5b).

The specificity of polyT CNA polymers was determined by mixing polyT CNA with complementary and non-complementary ssDNA (Figure 5c). PolyT CNA-2G polymers bound to polyA ssDNA (Figure 5c, Lane 2), but no binding was observed for a complete mismatch ssDNA (T10) (Figure 5c, Lane 6) or even a single base-pair mismatch ssDNA sequence (A5GA4) (Figure 5c, Lane10). These results confirmed the binding between CNA-2Gs and complementary ssDNA was sequence specific. For polyT CNA-1G polymers, on the other hand, no binding was observed even for the complementary ssDNA (Figure 5c, Lanes 3, 7, and 11). PolyT ssDNA was included as a positive control and also showed sequence specific binding (Figure 5c, Lanes 4, 8, and 12).

a)

b)

A10 1

2

3

4

5

6

7

8

9

10 11

12

1.0

Normalized intensity of unbound DNA band

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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CNAbound DNA Unbound DNA

0.5

R2 = 0.99

0.0 0 [CNA] (µM)

Kd ~ 70 µM

1

2

3

Log([CNA]) (µM)


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A10

c)

1

2

3

T10 4

5

6

7

A 5GA4 8

9

10

11 12

CNAbound DNA Unbound DNA

Figure 5. Gel electrophoresis evaluation of ssDNA hybridization with polyT CNA-2G (entry 5 from Table 1). a) Titration experiment showing increased binding of ssDNA (A10, 5 µM) with increasing concentration of CNA-2G polymers. A10 ssDNA = 5’-fluorescein-AAAAAAAAAA3’. Gel was run at 75 V for 155 min at 0 °C. b) Normalized intensity of the unbound DNA band was plotted against the concentration of loaded CNA-2G and the data were fit to a sigmoidal curve in GraphPad Prism. c) Gel electrophoresis experiment demonstrating the specificity of CNA-2G, but not CNA-1G, for complementary ssDNA. Lanes 1, 5, and 9 only contain A10 ssDNA (5 µM), T10 ssDNA (5 µM), and A5GA4 ssDNA (5 µM). Lanes 2, 6, and 10 contain CNA-2G (250 µM) with A10 ssDNA (5 µM), T10 ssDNA (5 µM), and A5GA4 ssDNA (5 µM), respectively. Lanes 3, 7, and 11 contain CNA-1G (250 µM) with A10 ssDNA (5 µM), T10 ssDNA (5 µM), and A5GA4 ssDNA (5 µM), respectively. Lanes 4, 8, and 12 contain polyT ssDNA (100 µM) with A10 ssDNA (5 µM), T10 ssDNA (5 µM), and A5GA4 ssDNA (5 µM), respectively. T10 ssDNA = 5’-TTTTTTTTTT-fluorescein-3’. A5GA4 ssDNA = 5’-fluoresceinAAAAAGAAAA-3’. The gel was run at 75 V for 165 min at 0 °C.


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CNA/DNA hybridization on nanoparticle surfaces. CNA-2G/ssDNA interactions were also examined on the surface of nanoparticles as a means for directing the assembly of the nanoparticles to aggregate structures (Figure 6). CdS-CNA nanoparticles that were prepared according to previous procedures,11,15 along with a DNA-A5 linker and DNA-complementary linker, were employed and expected to assemble in a manner illustrated in Figure 6a. In the scenario without DNA-A5 linker and DNA-complementary linker, CdS-CNA nanoparticles well dispersed in DMSO/water solution and examined by transmission electron microscopy (TEM) (Figure 6b). While, in the presence of DNA-A5 linker and a DNA-complementary linker, a dense assembly of CdS-CNA nanoparticle hierarchical structure was observed through TEM, which provides strong evidence for the CNA-2G/ssDNA interactions (Figure 6c). This assembly of nanoparticles was observed macroscopically as well (Figure 6d). Upon annealing, a “precipitate” of CdS nanoparticle aggregates settled to the bottom of the microcentrifuge tube. Heating, however, disrupted the assembly of particles, which re-dispersed to give a transparent nanoparticle solution. Implementation of CNA/DNA programmable CdS nanoparticle assembly is a promising method for generating colloidal superstructures with well-ordered geometries, which has significant potential for applications in optics, electronics, magnetic storage, and biological labeling areas.16

a)

b)

c)


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d)

Figure 6. CNA-2G/ssDNA induced nanoparticles assemblies. All the CdS-CNA nanoparticles and DNA linkers were annealed from 60 °C to room temperature in 500mM NaCl DMSO/H2O solution. a) Scheme for the self-assembly of CdS-CNA nanoparticles via interactions with DNAA5 linker, and DNA-complementary linker. b) TEM image of CdS-CNA nanoparticles after annealing without either DNA-A5 linker or DNA-complementary linker. c) TEM image of CdSCNA nanoparticles after annealing with DNA-A5 linker and DNA-complementary linker in a ratio of 4/1/1 (CdS-CNA/DNA-A5/DNA-complementary). d) A clear solution of CdS-CNA nanoparticles and DNA linkers was annealed from 60 °C to 4 °C at a rate of 1 °C/10 min. Yellow precipitates (nanoparticle aggregations) were observed, which re-dissolved after being heated at 60 °C for 15 min. CONCLUSION An extremely efficient thiol-ene photopolymerization was achieved to provide us a new generation of CNA polymers with a polythioether backbone and repeat unit length of 6 atoms, consistent with the spacing in native DNA. Highly selective hybridization between these CNA-


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2G polymers and complementary ssDNAs was observed and characterized via gel electrophoresis. Moreover, sequence specific bindings between CNA-2Gs and ssDNAs on the surface of nanoparticles were shown to induce the directed particle self-assembly. The versatility of this new generation of CNA, along with the relative ease and scalability of synthesis, portend the application of the artificial nucleic acid across a wide variety of research fields.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI:

The supporting information (SI) contains the characteristic data and NMR spectra of intermediates for CNA-2G monomer synthesis, CNA-2G monomer, and CNA-2G polymers; synthesis procedure and characteristic data for CNA-2G disulfide dimer; SEC traces for CNA2G monomer, acetyl protected CNA-2G monomer, TCEP protected CNA-2G monomer, and CNA-2G disulfide dimer; CNA concentration standard curve; Synthesis procedure for 10 nm CdS nanoparticles.

ACKNOWLEDGMENT We gratefully acknowledge financial support from NSF-MRSEC (DMR1420736).

REFERENCES (1) Watson, J. D.; Crick, F. H. C. The Structure of DNA. Nature 1953, 171, 737-738.


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New Generation of Clickable Nucleic Acids: Synthesis and Active

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